Chemical imaging combines optical spectroscopy and digital imaging for the molecular-specific analysis of materials. Raman, visible, near infrared (VIS/NIR) and Fluorescence chemical imaging have traditionally been performed in laboratory settings using research-grade light microscope technology as the image gathering platform. However, chemical imaging is applicable to in situ industrial process monitoring and in vivo clinical analysis. The application of chemical imaging outside the research laboratory has been limited by the lack of availability of stable imaging platforms that are compatible with the physical demands of industrial process monitoring and clinical environments. Both industrial and clinical settings often require compact, lightweight instrumentation suitable for the examination of remote areas that are inaccessible to conventional chemical imaging instrumentation and involve harsh chemicals in hostile areas. In addition, for in vivo cardio-vascular clinical applications, the presence of blood and bodily fluids limits the viewing, identification and ability to perform in vivo optical measurements of suspect areas.
Raman spectroscopy is one of the analytical technique that is broadly applicable and can be used for chemical imaging. Among its many desirable characteristics, Raman spectroscopy is compatible with samples in aqueous environments and can be performed on samples undergoing little or no sample preparation. The technique is particularly attractive for remote analysis via the use of optical fibers. By employing optical fibers for light delivery and collection the light source and light detector can be physically separated from the sample. This remote attribute is particularly valuable in sensing and analysis of samples found in industrial process environments and living subjects.
In a typical fiber-optic based Raman analysis configuration, one or more illumination fiber-optics deliver light from a light source (typically a laser) through a laser bandpass optical filter and onto a sample. The laser bandpass filter allows only the laser wavelength to pass while rejecting all other wavelengths. This purpose of the bandpass filter is to eliminate undesired wavelengths of light from reaching the sample. Upon interaction with the sample, much of the laser light is scattered at the same wavelength as the laser. However, a small portion of the scattered light (1 in 1 million scattered photons on average) is scattered at wavelengths different from the laser wavelength. This phenomenon is known as Raman scattering. The collective wavelengths generated from Raman scattering from a sample are unique to the chemistry of that sample. The unique wavelengths provide a fingerprint for the material and are graphically represented in the form of a spectrum. The Raman scattered light generated by the laser/sample interaction is then gathered using collection optics which directs the light through laser rejection filter which eliminates the laser light, allowing only Raman light to be transmitted. The transmitted light is then coupled to a detection system via one or more collection fiber-optics.
Previously described Raman fiber optic probe devices have several limitations. First, current fiber-optic-based Raman probes are sensitive to environmental variability. These devices often fail to function properly when the probe is subjected to hot, humid and/or corrosive environments. Several fundamental differences from current devices have been incorporated into the chemical imaging fiberscope design described here that address the environmental sensitivity issue. First, an outer jacket (or housing) that is mechanically rugged and resistant to varying temperatures and high humidity has been incorporated into the fiberscope design. Second, an optically transparent window that withstands harsh operating environment has been built into the probe at the fiberscope/sample interface. Normally, incorporation of a window into a probe would introduce a significant engineering problem. As emitted illumination light passes through the window and onto the sample, a portion of this light is back reflected by the window's inner and outer surfaces. In the prior art, this undesired back reflected light is inadvertently introduced into the collection fibers along with the desired Raman scattered light. The back reflected light corrupts the quality of the analysis. This problem is addressed in the current design by careful engineering of the aperture of the collection bundle taking into account the numerical apertures (NA) associated with the collection bundle fibers and collection lenses.
Previous probe designs are also inadequate because of the environmental sensitivity of the spectral filters that are employed in the devices. The chemical imaging fiberscope design of the current disclosure relies on spectral filter technologies that are remarkably immune to temperature and humidity. Past spectral filters have traditionally been fabricated using conventional thin film dielectric filter technology which are susceptible to temperature and humidity induced degradation in the filter spectral performance. The spectral filters described in the present disclosure employ highly uniform, metal oxide thin film coating material such as SiO2 which exhibits a temperature dependent spectral band shift coefficient an order of magnitude less than conventional filter materials. The improved quality and temperature drift performance of metal oxide filters imparts dramatically improved environmental stability and improved Raman performance under extreme conditions of temperature and humidity.
Another limitation of current probe technologies is that none combine the three basic functions of the chemical imaging fiberscope: (1) video inspection; (2) spectral analysis; and (3) chemical image analysis in an integrated, compact device.
Raman chemical imaging integrates the molecular analysis capabilities of Raman spectroscopy with image acquisition through the use of electronically tunable imaging spectrometers. In Raman chemical imaging, scattered Raman light is shifted in wavelength from the wavelength of the illuminating light. For example, Raman illumination at 532 nm can excite molecular vibrations in the sample at for example, 4000 cm−1 to produce scatter Raman light at lower and higher wavelengths of 439.3 nm and 647.5 nm, respectively. The Raman wavelength can be in the range of −4000–4000 cm−1. This produced Raman features 4000 cm−1 above the illuminating wavelength. Several imaging spectrometers have been employed for Raman chemical imaging, including acousto-optical tunable filters (AOTFs) and liquid crystal tunable filters (LCTFs). For Raman imaging, LCTFs are clearly the instrument of choice based on the following demonstrated figures of merit: spatial resolving power (250 nm); spectral resolving power (<0.1 cm−1); large clear aperture (20 mm); and free spectral range (0–4000 cm−1). LCTF's can also be designed by those skilled in the art to operate over different ranges of detection wavelengths that depend on the application from, for example, 400–720 nm, 650–1100 nm, 850–1800 nm or 1200–2400 nm. AOTFs and LCTFs are competitive technologies. AOTFs suffer from image artifacts and instability when subjected to temperature changes.
Under normal Raman imaging operation, LCTFs allow Raman images of samples to be recorded at discrete wavelengths (energies). A spectrum is generated corresponding to thousands of spatial locations at the sample surface by tuning the LCTF over a range of wavelengths and collecting images systemically. Contract is generated in the images based on the relative amounts of Raman scatter or other optical phenomena such as luminescence that is generated by the different species located throughout the sample. Since a spectrum is generated for each pixel location, chemometric analysis tools such as Cosine Correlation Analysis (CCA), Principle Component Analysis (PCA) and Multivariate Curve Resolution (MCR) are applied to the image data to extract pertinent information.
Chemical imaging can be performed not only in a scattering mode at high resolution as done for Raman chemical imaging using laser illumination, but it can also be conducted for broadband incident illumination (wavelength>10 cm−1) at corresponding reduced spectral resolution (wavelength>10 cm−1). This broadband illumination and reduced resolution spectroscopy can be done in the UV wavelength (200–400 nm), VIS wavelength (400–780 nm) and NIR wavelength (780–2500 nm) regions to measure the optical absorption and emission from the sample. Performing such absorption or emission measurements using a fiberscope requires addressing many of the same problems as encountered in performing Raman imaging. The ability to perform combinations of these optical measurements and chemical imaging in the same fiberscope system is also an advantage in that enabling different chemical imaging technologies in one platform provides valuable complementary information.
One problem in performing chemical analysis and chemical imaging in the human body, such as in for example, in the cardio-vascular system or body cavities during, for example, endoscopic surgery, is the occurrence of significant amounts of blood and water at the sample site which both scatters and absorb light in certain wavelength ranges. Further, the positioning of a fiberscope probe to perform an in vivo optical analysis requires accurate steering and viewing thru these body fluids so as to define regions of interest and accurately position the optical probe at the region to be sampled. Viewing more than a few millimeters through blood requires observation at NIR wavelengths. However, such NIR wavelengths are poorly suited for performing Raman scattering or fluorescence measurements.
For example, identification and characterization of vulnerable plaque in the cardio-vascular system is critically related to Cardio vascular disease which is a leading cause of deaths in the United Stated. The in vivo identification and characterization of plaques in the cardio vascular system requires locating the suspect regions and positioning a sampling probe to analyze these regions. Other current methods for characterizing vulnerable plaque such as Intra Vascular UltraSound (IVUS) and thermometry (e.g., Volcano Therapuetics, Inc.) map out some physical properties of the arterial walls to suggest likely areas of plaques, but are not chemically specific and cannot provide any detailed analytical information regarding the chemical state or molecular composition of these target areas or plaques. Optical imaging to position a chemical probe in vivo is desirable but problematic and limited due to the scattering and absorption properties of blood. While certain optical wavelengths in the NIR are known to be more favorable than others for in vivo viewing of the cardiovascular system, these wavelengths are not well-suitable for performing highly specific chemical analysis. For example, the Raman scattering cross sections at longer wavelengths (e.g., NIR) are reduced from VIS wavelength excitation by the fourth power of their respective frequencies. The low cost, high sensitivity Si charge-coupled detectors (“CCD”) used for Raman Chemical imaging also have reduced sensitivity for longer wavelength Raman scattered peaks thereby making it difficult to detect the very important CH-bond vibrational region.
Thus, there is a need for an apparatus and method to enable long range viewing, steering and targeting which is optimal in the NIR as well as subsequent and/or simultaneous chemical imaging of the target area which is optimal in the visible range. This invention addresses that need.